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Abstract

Nucleosomes are the fundamental unit of eukaryotic chromosome packaging, comprised of 147 bp of DNA wrapped around two molecules of each of the core histone proteins H2A, H2B, H3, and H4. Nucleosomes are symmetrical, with one axis of symmetry centered on the homodimeric interaction between the C-termini of the H3 molecules. To explore the functional consequences of nucleosome symmetry, we designed an obligate pair of H3 heterodimers, termed H3X and H3Y, allowing us to compare cells with single or double H3 alterations. Our biochemical validation of the heterodimeric X-Y interaction included intra-nucleosomal H3 crosslinking using dimethyl suberimidate (DMS). Here, we provide a detailed protocol for the use of DMS to analyze yeast nucleosomes.

Post-translational modifications of histone proteins affect every aspect of chromosome biology, including transcription, replication, repair, and recombination. Because nucleosomes contain two copies of each core histone, modifications could be symmetric (same modifications on both H3 tails, e.g., K27me on both H3 tails within a nucleosome (Voigt et al., 2012)) or asymmetric (modifications on a single H3 tail, e.g., K27me on a single H3 tail within a nucleosome (Voigt et al., 2012)). Recent studies have demonstrated that nucleosomes in mammalian cells indeed display some asymmetric modifications (Voigt et al., 2012; Shema et al., 2016). To allow experimental manipulation of nucleosomal symmetry in vivo, we designed a pair of altered histone H3 proteins that have obligate heterodimeric interactions, termed H3X (L126A, L130V) and H3Y (L109I, A110W, L130I) (Ichikawa et al., 2017). Yeast cells expressing both H3X and H3Y are viable, but inviable if cells express only H3X or H3Y.

For biochemical validation of H3X-H3Y interactions within individual nucleosomes, we generated yeast strains expressing the bacterial biotin ligase BirA, N-terminal V5-tagged H3X and N-terminal biotin-accepting epitope tagged H3Y (Beckett et al., 1999). BirA is an enzyme that attaches biotin to a specific acceptor epitope, enabling us to purify the biotinylated molecules by streptavidin affinity chromatography. We treated extracts from yeast cells with dimethyl suberimidate (DMS), a crosslinking agent that contains a primary amine reactive imidoester group at each end of an 8-atom spacer arm (Figure 1A). DMS produces well-characterized crosslinks within histone octamers, including links between the two H3 molecules (Figure 1B; Kornberg and Thomas, 1974; Thomas, 1989). Therefore, this method can be used to report on the composition of asymmetric epitope tags.

Crosslinked samples are digested with micrococcal nuclease (MNase) to generate a soluble population of chromatin fragments containing approximately mononucleosome-sized DNA molecules (we note that DMS crosslinking prevents generation of a uniform ladder of MNase-digested products, Figure 1C). Biotin-tagged, MNase-digested chromatin is then purified via streptavidin-agarose affinity purification in the presence of high salt (2 M NaCl). This salt concentration is sufficient to remove DNA from histones (Bartley and Chalkley, 1972), avoiding interference from any neighbor nucleosomes that survived the MNase digestion. Bound proteins are then analyzed by Western blotting (Figure 1D). The DMS crosslinking efficiency of the X-Y heterodimeric pairs was around 10%, nearly identical to wild-type H3 homodimeric pairs; additionally, approximately 20% of the crosslinked heterodimers in the input fractions were precipitated by streptavidin-agarose (Ichikawa et al., 2017). We applied this method to analyze the extent of homodimerization of H3X or H3Y, as well as X-Y heterodimerization (Ichikawa et al., 2017). To examine this, we quantified X-Y dimer bands rather than the monomer, because these DMS crosslinked species represent direct H3-H3 interactions within individual nucleosomes.

Figure 1. Biochemical validation of asymmetric nucleosome formation in vivo. A. Chemistry of DMS cross-linking. DMS reacts with primary amines of proteins to form amidine bonds. B. Schematic for DMS crosslink of H3X and H3Y heterodimer. Yeast strains expressed V5-tagged H3X and Biotin-tagged H3Y, as indicated. C. DNA samples purified from MNase-digested chromatin from each time point (0, 10, 20 min) were analyzed by electrophoresis on a 1.5% TAE agarose gel, and stained with ethidium bromide. Note that after DMS crosslinking, the MNase-digested DNA fragments do not display the characteristic polynucleosomal ladder of uncrosslinked chromatin. D. Immunoblot analysis of V5-H3X and biotin-H3Y interactions. The left two lanes show total uncrosslinked and DMS-crosslinked chromatin, and right lanes show MNase-digested chromatin (Input), flow through fraction (Unbound) and streptavidin-precipitated biotinylated-H3 (Bound). Samples were separated by 17% SDS-PAGE, transferred to a membrane, and probed with anti-V5 antibody.

Day 1
Pick a single colony from a plate, and inoculate an overnight culture of cells in 15 ml of synthetic media. Grow at 30 °C, 170 rpm in a shaker incubator. Do this 1-2 days beforehand, depending on growth rate of strain.

Day 2
Inoculate the overnight culture grown on day 1 into 110 ml of synthetic media supplemented with 250 nM biotin. Biotin is added to favor in vivo biotinylation of the tagged H3 proteins. The amount of cells to inoculate depends on growth rate (see below; typically, inoculate 3 OD units of cells of most X-Y strains into 110 ml media, which are then grown for 12 h). Grow at 30 °C, 170 rpm in a shaker incubator.

Day3

Cross-linking H3 dimers with DMS

Harvest at desired cell density. The desired cell density is 0.25 at OD600; don’t use more than 100 ml of cells at OD600 = 0.3 per Streptavidin-pull down described below, in order to assure that chromosomes are adequately digested with 20 μl MNase to generate mostly monosomes.

Wash 3 times with 1 ml E buffer by vortex. Spin down in microfuge 20 sec at max speed at 4 °C. Remove supernatant. Thorough washing is important here to remove amine-containing compounds that will impair crosslinking.

Add 1/100 volume of 1 M MgCl2 to a final concentration of 10 mM (really 8 mM final, since E buffer contains 2 mM EDTA), and add 1/100 volume of 0.1 M CaCl2 to a final concentration of 1 mM to the DMS-crosslinked sample (the remaining amount after Step A-13, approximately 800 μl) at room temperature. Take 100 μl to generate an un-digested DNA sample for gel analysis. Store on ice.

Equilibrate at 37 °C in a water bath for 5 min.

Add 20 μl of MNase, invert the tubes 5 times and incubate at 37 °C for 20 min.

Add 1/25 volume of 0.25 M EDTA/EGTA to a final concentration of 10 mM and invert 5-10 times to inhibit MNase. Take 100 μl to generate an MNase-digested DNA sample. Spin at 8,000 x g for 1 min, 4 °C, take supernatant for Streptavidin-pull down.

Gel analysis of MNase digestion

For DNA purification, add 5 μl of RNase A (10 mg/ml) to the 100 μl sample aliquots and incubate at 37 °C for 30 min.

Wash beads three times with 1 ml W buffer at 4 °C, by rotating at 4 °C for 5 min each time. Spin at 8,000 x g for 1 min at 4 °C, then discard supernatant.

After the last wash, remove all supernatant with a 26 gauge needle on a vacuum. Resuspend the beads in 50 μl 2x SB. Store at -20 °C.

Western blot

Boil samples at 100 °C for 10 min.

Load 10 μl of 0 min and 60 min samples, 15 μl of Input samples and 8 μl of Bound samples. Run on 17% SDS-PAGE gels in 1x SDS-running buffer at constant 8 mA while bromophenol blue goes through the stacking gel, and 18 mA as it goes through the resolving gel. Continue running for 15 min after bromophenol blue runs off the bottom to improve the resolution of histones. Total run time is approximately 2 h.

Transfer to Nitrocellulose membrane in blotting buffer at constant 0.5 A for 48 min.

Remove the TBST and incubate the membrane with 1 ml ECL (enhanced chemiluminescence) solution for 10 min. Detect by using ChemiDoc Touch Imaging System (Chemiluminescent Blot mode, at the highest resolution).

Data analysis

H3-H3 crosslinked species were quantified with Bio-Rad ‘Image Lab’ software, using the ‘Volume Tools’. The area of the band was defined by surrounding it with a rectangle box (Figure 3). The same volume area was used to measure background signals, which were subtracted from the band intensity. The percentage of precipitated H3 dimer was calculated with following formulas:
Total input = ‘Band intensity of H3 dimer on input lane’ x ‘Total volume of input fraction’/‘Loading volume of input fraction’
Total bound = ‘Band intensity of H3 dimer on bound lane’ x ‘Total volume of bound fraction’/‘Loading volume of bound fraction’
Percentage of precipitated H3 dimer = (Total bound/Total input) x 100
The mean and standard deviation were calculated from the values of 3 independent replicate experiments.

Figure 3. Data analysis with Bio-Rad image Lab. Areas of the H3-H3 dimer bands and the blanks were defined by surrounding it with a rectangle box using the ‘Volume Tools’.

Notes

Use fresh cells (don’t freeze cells before the crosslink).

Use fresh DMS stock.

Note that during bead beating, visible aggregates appear, and many of these remain in the screwcap tube with the discarded glass beads at Step A7 of the DMS cross-link section. This is normal and does not indicate a problem.

60 min of incubation during the DMS crosslinking reaction at Step A11 was sufficient for us to obtain crosslinked H3 dimers. However, you can change the incubation time depending on your purpose and sample conditions.

PVDF membranes can be used for the Western blot, but in our experience Nitrocellulose membranes detect histones more sensitively.

Acrylamide is toxic. Wear examination gloves when you make SDS-PAGE gels.